This proposal focuses on incorporating protein flexibility into drug discovery by using ensembles of protein conformations (multiple protein structures, MPS) to represent inherent flexibility. This approach has been shown to overcome some limitations of traditional docking to rigid structures, resulting in higher hit rates and greater chemical diversity of identified inhibitors. More importantly, the current aims evolve the idea that a protein's conformational behavior can be used to identify new modes of inhibition. The long-term goal of this work is to improve the field of structure-based drug discovery (SBDD) by developing methods that more accurately model target proteins and incorporate the vast information available from structural proteomics. This study is well integrated, providing both methodological development and practical application to systems of critical biomedical importance to prove overall utility of the techniques. The first aim (SA1) examines various improvements to the MPS methodology. Alternative sources of MPS will be used. Mixed solvent simulations are proposed to enhance mapping the protein surface. Benchmark data from multiple solvent crystal structures will identify which algorithmic strategies perform best across many proteins. Applicability to allosteric sites will be examined. SA2 and SA3 conduct computational studies of protein dynamics to drive the discovery of new inhibitors for HIV-1 protease (HIVp) and b-secretase (BACE1), respectively. Both proteins are aspartyl proteases, and their large degree of flexibility greatly affects ligand binding and inhibition. The MPS approach has proven advantageous for systems with large, exposed binding sites that are problematic for traditional docking. Targeting new modes of inhibition for HIVp has the promise of reducing drug resistance in AIDS treatment. Our pursuit of alternative modes of inhibiting BACE1 will focus on identifying smaller lead compounds that are more likely to cross the blood-brain barrier; this pharmacokinetic property is absolutely essential to treat Alzheimer's disease but is lacking in most inhibitors in the literature. Experimental verification of the MPS methodology is a key component of the later aims, including assaying potential inhibitors and performing key structural studies by deuterium exchange, crystallography, and NMR.